Showing papers on "Melibiose published in 1996"
TL;DR: The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the α-galactosidase previously purified from T. reesei, and the deduced amino acid sequences of A GLI and AGLIII showed similarity with the β-mannosidases of plant, animal, yeast and filamentous fungal origin.
Abstract: Three α-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevislae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside. The genes agll, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the α-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLIII showed similarity with the bacterial α-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-α-d-galactopyran-oside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galac-tose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-l,4-β-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-β-mannanase of T. reesei and a β-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the α-galactosidase previously purified from T. reesei.
90 citations
TL;DR: The findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane.
Abstract: In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity. In general agreement with a previously suggested secondary structure model of melibiose permease [Botfield, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T.H. (1992) J. Biol. Chem. 267, 1818], clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops. Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane. Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937], the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments. However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.
79 citations
TL;DR: It is suggested that lactose may be considered as an appropriate starting material for synthesizing large amounts of a trisaccharide that effectively neutralizes anti-Gal.
Abstract: Rejection of pig-to-human or pig-to-primate xenografts is mediated by the natural anti-Gal antibody, which interacts with alpha-galactosyl epitopes (i.e., Gal alpha1-3Gal beta1-4GlcNAc-R) abundantly expressed on porcine cells. The objective of this study was to determine the ability of various synthetic oligosaccharides to inhibit the binding of anti-Gal IgG molecules to porcine endothelial cells in vitro. Such inhibition ultimately may help to reduce or to prevent the in vivo antibody-dependent cell cytotoxicity (ADCC) reaction. In the absence of complement-mediated hyperacute rejection, the ADCC induced by anti-Gal IgG molecules is likely to cause the chronic rejection of xenografts. The synthetic free alpha-galactosyl epitope (Gal alpha1-3Gal beta1-4GlcNAc) was found to be 300-fold more effective than melibiose or alpha-methyl galactoside in inhibiting anti-Gal binding to porcine endothelial cells, and to prevent >90% of the antibody binding at a concentration of 1 mM. The disaccharide Gal alpha1-3Gal was ten-fold less effective than the free alpha-galactosyl epitope. Accordingly, the affinity of the disaccharide to anti-Gal, as measured by equilibrium dialysis, was seven-fold lower than that of the trisaccharide. The effective concentration of oligosaccharides inhibiting anti-Gal is independent of the antibody affinity, but is dependent on the concentration of the antibody. Based on the small difference in affinity between Gal alpha1-3Gal beta1-4GlcNAc and Gal alpha1-3Gal beta1-4GIc, and the large difference in the price of N-acetyllactosamine vs. lactose, it is suggested that lactose may be considered as an appropriate starting material for synthesizing large amounts of a trisaccharide that effectively neutralizes anti-Gal.
59 citations
TL;DR: The results substantiate the conclusion that regulation of the lactose permease in E. coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.
Abstract: Allosteric regulation of several sugar transport systems such as those specific for lactose, maltose and melibiose in Escherichia coli (inducer exclusion) is mediated by the glucose-specific enzyme IIA (IIAGlc) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Deletion mutations in the cytoplasmic N and C termini of the lactose permease protein, LacY, and replacement of all cysteine residues in LacY with other residues did not prevent IIAGlc-mediated inhibition of lactose uptake, but several point and insertional mutations in the central cytoplasmic loop of this permease abolished transport regulation and IIAGlc binding. The results substantiate the conclusion that regulation of the lactose permease in E. coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.
29 citations
TL;DR: The hydrolysis inhibition in the presence of a substrate has been shown to correlate with the substrate transglycosylation, and data of steady- state kinetics together with data of presteady-state kinetics obtained by the stop-flow method suggest that an intermediate galactosyl-enzyme complex is formed in the reaction and is of particular importance in the processes under study.
29 citations
TL;DR: Direct evidence is presented that the multiple-sugar metabolism genes can be transcribed as a single operon in Streptococcus mutans.
Abstract: The multiple-sugar metabolism (msm) locus of Streptococcus mutans constitutes a non-PTS sugar uptake system responsible for the transport and utilization of raffinose, melibiose and isomaltotrioses. While previous studies have used polar mutations to suggest that these genes are co-transcribed, there has not been any direct evidence to support this. In this report we present direct evidence that the msm genes can be transcribed as a single operon.
25 citations
TL;DR: The characterization of the SNF1 homolog of Candida glabrata, a pathogenic yeast phylogenetically related to S. cerevisiae, resulted in the loss of the ability to utilize trehalose, indicating that even in an organism with such a limited carbon utilization spectrum, the regulatory mechanism governing catabolic repression is preserved.
Abstract: The SNF1 gene product, a serine/threonine protein kinase, is a global regulatory protein which has been isolated from several organisms. In Saccharomyces cerevisiae the SNF1 gene product is essential for the derepression of glucose repression since snf1 strains are unable to utilize sucrose, galactose, maltose, melibiose, or nonfermentable carbohydrates. Moreover, the SNF1 gene product was suggested to interact with additional regulatory pathways and to affect the expression of multiple target genes as reflected by the pleiotropic nature of the snf1 mutation. Here we report the characterization of the SNF1 homolog of Candida glabrata, a pathogenic yeast phylogenetically related to S. cerevisiae. The carbon utilization spectrum of C. glabrata is considerably narrower than that of other pathogenic yeasts, and the majority of the strains utilize solely glucose and trehalose from among 20 of the most commonly tested carbohydrates. Disruption of the C. glabrata SNF1 homolog resulted in the loss of the ability to utilize trehalose, indicating that even in an organism with such a limited carbon utilization spectrum, the regulatory mechanism governing catabolic repression is preserved.
23 citations
TL;DR: It was concluded that there is some type of physiological interaction between His-322 and Glu-325 and that the complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a newnegative charge at 322.
22 citations
TL;DR: The results suggest that Asn-58 is important for Na+ recognition and that Leu-54/Ala-58, a double mutant, shows moderate sugar accumulation with cation selectivity similar to wild-type.
21 citations
TL;DR: Stachyose was the most effective inducer, followed by melibiose, raffinose, and galactose; scarce activity was detected with glucose; the addition of cyclic adenosine-3′,5′ monophosphate (cAMP) suggests that this compound could be involved in the regulation of α-gal synthesis.
Abstract: The influence of soybean galactosaccharides (stachyose, raffinose, melibiose) as well as galactose and glucose on the synthesis and activity of alpha-galactosidase (alpha-gal) from Lactobacillus fermentum CRL 251 was studied. Stachyose was the most effective inducer, followed by melibiose, raffinose, and galactose; scarce activity was detected with glucose. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of L. fermentum growing on galactose. This effect was reversed by the addition of cyclic adenosine-3',5' monophosphate (cAMP), which suggests that this compound could be involved in the regulation of alpha-gal synthesis.
18 citations
TL;DR: The yeast Torulaspora delbrueckii IFO 1255 was selected as the strain fermenting melibiose from 35 strains of Torulasporpora species as discussed by the authors, which produced extracellular and cell-associated forms of α-galactosidase when grown on either melibioses or galactose as the sole carbon source.
Abstract: The yeast Torulaspora delbrueckii IFO 1255 was selected as the strain fermenting melibiose from 35 strains of Torulaspora species. The strain IFO 1255 produced extracellular and cell-associated forms of α-galactosidase when grown on either melibiose or galactose as the sole carbon source. Most of the enzyme was located outside of the cell membrane: the periplasmic space, or cell walls, or both. α-Galactosidase was purified to homogeneity from the cell-free extract of the strain IFO 1255 by acid treatment and column chromatography on DEAE-Toyopearl 650M and Butyl-Toyopearl 650M. The molecular weight of the purified enzyme was estimated to be 88 000 by SDS-polyacrylamide gel electrophoresis and 530 000 by gel filtration. The enzyme contained 50% of its molecular weight as carbohydrate. Optimum pH and temperature were 4.5–5.5 and 55°C, respectively. The enzyme was inhibited strongly by Ag2+, Hg2+ and Cu2+ each at 1 mmol 1-1. The Km (μmol 1-1) for p-, o-, m-nitrophenyl α-D-galactopyranoside, melibiose, raffinose and stachyose were 2.8, 1.3, 2.8, 4.2, 170 and 230, respectively, and Vmax (μmol min-1 mg protein-1) for those substrates were 310, 140, 21, 22, 30 and 44, respectively. The properties of α-galactosidase from T. delbrueckii IFO 1255 were similar to those from the related species, Saccharomyces cerevisiae.
TL;DR: Experiments indicate that the model is able to accurately predict the cell mass profiles for yeast growth, which can resemble profiles for growth on a single substrate (melibiose) or can resemble diauxie growth.
Abstract: Cybernetic modeling has traditionally been used in the modeling of microbial growth on multiple substrates. In this paper, cybernetic modeling has been applied to serve as a model for growth on substrates such as melibiose, which are disaccharides and enzymatically degrade to a monosaccharide mixture in the fermentation broth. The enzyme alpha-galactosidase has been shown to be strongly induced in the presence of galactose and severely repressed by glucose. In the present model, the relative concentration of alpha-galactosidase has been linked to that of the key enzyme for galactose metabolism. The enzymatic degradation process is placed under the control of the cybernetic variables. The maximum rate of melibiose degradation vm and the Monod parameters for growth of Saccharomyces cerevisiae on pure glucose and galactose were estimated by batch growth experiments. S. cerevisiae growth on melibiose and a mixture of melibiose and glucose under a variety of preculturing conditions was simulated. Depending on the rate of enzymatic degradation (i.e., the value of vm), the cell mass profile for microbial growth on a disaccharide can resemble profiles for growth on a single substrate (melibiose) or can resemble diauxie growth. Experiments indicate that the model is able to accurately predict the cell mass profiles for yeast growth.
05 Feb 1996
01 Jan 1996
TL;DR: Several oligosaccharide-substituted polymers of which chemical structures are shown are of interest as biological probes and cell-specific biomedical materials are prepared.
Abstract: Well-defined polymers having mono- and oligosaccharide chains are of interest as biological probes and cell-specific biomedical materials. We prepared several oligosaccharide-substituted polymers of which chemical structures are shown in the next page. The main chains of the polymers are the derivatives of polystyrene (1–11, 18–21), Polyacrylamide (12–17), polypeptide (22), and polysaccharide (23). The starting mono- and oligosaccharides include glucose (1,12,21), mannose (14,23), N-acetylglucosamine (13), cellobiose (3), maltose (2), maltotriose (6), maltopentaose (7), maltoheptaose (8), amylose (9), lactose (4,16,17,19,20,22), melibiose (5), N-acetyllactosamine (15), N,N′-diacetylchitobiose(18), N,N′,N″-triacetylchitotriose(11), and so on.